Structural basis and specificity of human otubain 1-mediated deubiquitination.
OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.
Edelmann
Mariola J
MJ
Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX37BN, UK.
Iphöfer
Alexander
A
Akutsu
Masato
M
Altun
Mikael
M
di Gleria
Katalin
K
Kramer
Holger B
HB
Fiebiger
Edda
E
Dhe-Paganon
Sirano
S
Kessler
Benedikt M
BM
D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D000072017:Deubiquitinating Enzymes; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry; D014452:Ubiquitins / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology
D014452:Ubiquitins; C496399:OTUB2 protein, human; D013869:Thiolester Hydrolases; D010450:Endopeptidases; D000072017:Deubiquitinating Enzymes; C538826:OTUB1 protein, human; D003546:Cysteine Endopeptidases